This EUREKA proposal addresses two interlocked questions on neurovascular coupling: 1 - What are the design principles that link cortical blood flow dynamics to the underlying angioachitecture? In particular, is the microvasculature arranged in modules or does it form a continuum? 2 - What are the signaling mechanisms that neurons use to transform electrical activity into changes - both increases and decreases - in blood flow? In particular, is the relation between metabolism and blood flow mediated by vasoactive signaling molecules under the control of inhibitory interneurons? These are central questions in brain science, with many innovative hypotheses on the regulation and control of arterial transport. However, a dearth of experimental approaches has limited the formation of a central dogma on a vascular unit and the control of blood in the brain. Future progress will depend on the strong interplay of concepts from physics, neurophysiology, molecular biology, and experimental neurology. The confluence of this interplay, an essential feature of the proposed studies, should delineate emergent properties of vascular networks. Our approach makes use of four unique and in some cases developing tools: (1) "All Optical Histology" and associated computational procedures to reconstruct the angioarchitecture and cytoarchitecture of the mouse brain. These studies address the structure of the microvasculature. (2) Perturbation techniques, mediated by linear and nonlinear optical interactions, to modulate blood flow in targeted vessels. These studies probe the redundancy of flow, test models of experimental microstroke, and provide one means to study the consequence of changes in flow on neuronal activity. (3) Sentinel cells, formed by transforming HEK cells to respond to exogenous transmitters with an optical read-out, to directly measure patterns of vasoactive molecules. (4) In vivo two-photon guided patch to record from and stimulate inhibitory interneurons that release vasoactive substances. In combination with two photon measurements of blood flow, we can determine quantitative relations between neuronal activity and blood flow. These proposed questions bear on fundamental issues of blood flow in the normal brain including homeostasis and the underpinnings of blood-based imaging techniques such as fMRI - and issues in dysfunctional states - such as microstroke and microvascular diseases. PUBLIC HEALTH RELEVANCE: Uninterrupted blood flow in the brain is essential to maintain all aspects of cognition as well as homeostasis of bodily functions. The proposed work may significantly increase our understanding of how critical flow is regulated in both normal and dysfunctional states. Such understanding bears directly on the interpretation of diagnostics to monitor basal flow and the changes in flow that are induced by mental activity. Further, our work may suggest pathways for the potential treatment of microstroke and microvascular diseases.